An Overview of In Vitro Genotoxicity Testing Strategies for Fragrance Materials: Addressing False Positives and Advancing Non-Animal Approaches.

The European Union's ban on in vivo genotoxicity testing for cosmetic ingredients (Directive 2003/15/EC), followed by the Scientific Committee on Consumer Safety (SCCS) 7th amendment, has created an urgent need for robust, non-animal genotoxicity testing strategies. Standard regulatory in vitro assays - including the Ames test, the in vitro micronucleus test, and chromosomal aberration assays - are prone to high rates of misleading positive results (up to 93% for cosmetic ingredients), primarily because they lack sufficient phase II metabolic competence. This review describes a tiered, mechanism-informed, fully in vitro testing strategy that integrates: (1) the ToxTracker assay, a green fluorescent protein (GFP)-reporter stem cell platform for mechanistic follow-up; (2) the 3D Reconstructed Skin Micronucleus (RSMN) assay, which provides a physiologically relevant dermal exposure model; (3) the Chicken Egg Genotoxicity Assay (CEGA) and Hen's Egg Test for Micronucleus Induction (HET-MN), which leverage the metabolic competence of fetal chicken systems; and (4) in silico metabolism prediction using OASIS TIMES. A proposed decision-making flow chart is provided. Two case studies, 2-octen-4-one and p-methoxycinnamaldehyde, illustrate how this strategy can resolve ambiguous in vitro findings and distinguish true genotoxic hazards from non-biologically relevant positives.